JouRNAL oF VnoLoGy, Dec. 1975, p. 1512-1527 Vol. 16, No. 6 Copyright ©) 1975 American Society for Microbiology Printed in U.SA. Replication of Picornaviruses I. Evidence from In Vitro RNA Synthesis that Poly(A) of the Poliovirus Genome is Genetically Coded KAROLINE DORSCH-HASLER, YOSHIAKI YOGO,' AND ECKARD WIMMER* Department ofMicrobiology, St. Louis University School ofMedicine, St. Louis, Missouri 63104, and Department ofMicrobiology, School ofBasic Health Sciences, State University ofNew York at Stony Brook, Stony Brook, New York 11794 * Received for publication 3 July 1975 A crude replication complex has been isolated from poliovirus-infected HeLa cells and used for synthesis of poliovirus replicative intermediate (RI) RNA, replicative form (RF) RNA, and single-stranded (SS) RNA in vitro. All three classes of virus-specific RNA synthesized in vitro are shown to contain poly(A). Poly(A) of RF and of SS RNA [RF-poly(A) and SS-poly(A)] has a chain length (50 to 70 nucleotides) that is shorter than that of poly(A) of in vivo-synthesized RNAs. Poly(A) of RI [RI-poly(A)], however, is at least 200 nucleotides long and, therefore, larger than poly(A) of RI isolated from HeLa cells 4 h after infection. The crude membrane-bound replication complex contains a terminal adenylate transferase activity that is stimulated by Mn2+ and the addition of an (Ap),AoH primer. This transferase activity is found also in extracts of mock-infected cells. Partial purification of the replication complex in a stepwise sucrose gradient, in which the viral replicase is associated with the smooth cytoplasmic membrane fraction, does not remove the terminal transferase. However, when the partially purified replication complex is treated with deoxycholate and sedimented through a sucrose gradient, a soluble replication complex can be isolated that is free from terminal adenylate transferase. This soluble replication complex was found to synthesize viral RNA-linked poly(A) longer in chain length than that synthesized by the crude replication complex. Taking into account the 5'- terminal poly(U) in poliovirus minus strands, our data suggest that polyadenyla- tion of poliovirus RNA occurs by transcription and not by end addition. When compared to other viral systems, poliovirus and, probably, all picornaviruses appear to be unique in that the poly(A) of their genome is genetically coded. Poly(A) covalently linked to mRNAs ofeukar- Mounting evidence suggests that poly(A) of yotic cells has been a conundrum ever since its cellular mRNA is synthesized by post-transcrip- discovery some years ago (for a review, see tional addition. Terminal adenylate transfer- reference 25). Its mode of biosynthesis is poorly ases, which are presumably involved in this understood and its biological function remains process, have been isolated from mammalian obscure. Poly(A) was originally thought to play cells and characterized (for a review, see refer- a crucial role in the synthesis of cellular ence 11). Terminal adenylate transferase has mRNA's and their transport to the cytoplasm. also been found in vaccinia virions (12, 30, 32, It was, furthermore, suggested that poly(A) 42). This enzyme is likely to polyadenylate viral might be a requirement for an mRNA to func- transcripts by end addition because vaccinia tion in protein synthesis. However, the lack of virus-specific mRNA contains poly(A) (22), poly(A) in a large proportion of mRNA's of whereas the viral genome does not contain HeLa cells (1, 29) and of sea urchin embryos (31) tracts ofpoly(dT) (32, 42). However, no evidence and the absence of poly(A) in all reovirus has been presented that directly links any of mRNA's (46) indicate that transcription, proc- the characterized terminal adenylate transfer- essing, transport, and translation of mRNA's ases with the poly(A) synthesis of mRNA. can occur in eukaryotic cells without the in- Single-stranded (SS) RNA viruses, which rep- volvement of poly(A). licate in the cytoplasm, have been classified I Present address: Department of Viral Infection, Insti- into plus-strand and minus-strand viruses (5). tute of Medical Science, Tokyo, 108 Japan. The genome RNAs of minus-strand viruses, 1512 VOL. 16, 1975 REPLICATION OF PICORNAVIRUSES 1513 such as rhabdoviruses, serve as templates in when compared with the other systems that primary transcription that precedes viral pro- have been analyzed so far. tein synthesis early in infection. Transcription (This work was presented in part at the 73rd can be carried out in vitro with detergent-dis- Annual Meeting of the American Society for rupted virions because of a virion-associated Microbiology, 6 to 11 May 1973, Miami Beach, RNA polymerase (7). Viral mRNA's isolated Fla.) from infected cells or synthesized in vitro are MATERIALS AND METHODS poly(A)-linked (40). The genome (template) RNA not contain of Cells and virus. The growth of strain S3 HeLa does segments poly(U) (18, cells in suspension culture and the propagation of 28), suggesting again post-transcriptional end poliovirus type 1 (Mahoney) have been described addition as the mechanism of poly(A) synthesis previously (50). (48). In contrast to vaccinia virus, however, no Preparation of the crude replication complex. poly(A) polymerase activity has been found so HeLa cells were washed once in Earle saline, sus- far to be associated with minus-strand virions pended at a concentration of4 x 107 cells/ml in Eagle (8). minimal essential medium without serum, and in- Picorna and togaviruses are plus-strand fected with 40 to 50 PFU of virus per cell. After RNA viruses, the single-stranded genome of adsorption at room temperature for 30 min, the cells which is polyadenylated and serves as mRNA were diluted to 4 x 106/ml with warm medium con- taining horse serum or heat-inactivated calf serum during initiation of infection (40). Poly(A) in (5%) and incubated at 37 C. We have found that the poliovirus RNA was discovered by Armstrong growth of HeLa cells and the virus yield were inde- et al. (2) and subsequently was shown by us to pendent of the serum used. The crude polio replica- be 3' terminal and heterogeneous in size with tion complex was isolated as follows (27). Cells were an average chain length of 89 adenylate resi- harvested 4 h after infection, washed once with dues (50). Earle saline, and suspended in reticulocyte standard We have studied the biosynthesis ofthe homo- buffer (RSB; 0.01 M Tris-hydrochloride, pH 7.35; polymeric tail of the poliovirus genome. Our 0.01 M KCl) to which MgCl2 had been added (to 1.5 strategy for solving the problem was the follow- mM) at a concentration of 2 x 107 cells/ml. After swelling for 5 min at 0 C, the cells were disrupted ing: first, we have analyzed the structure of with a stainless-steel Dounce homogenizer. Nuclei polio minus strands (that is, RNA complemen- and cell debris were removed by centrifugation at tary to the genome) for a sequence complemen- 600 x g for 5 min, and the replication complex was tary to poly(A). This led to the discovery of sedimented from the supernatant by centrifugation poly(U) in minus strands of polio double- at 30,000 x g for 20 min. The resulting pellet was stranded RNA (referred to as replicative form then suspended at approximately 10 mg of pro- [RF]; 51) and of the polio replicative intermedi- tein/ml in suspending buffer (0.12 M Tris-hydrochlo- ate (RI; 52) in the 5'-terminal position (49). ride, pH 8.0; 0.06 M KCl). Such preparations can be we have studied stored for several months at -70 C without loss of Second, the synthesis of polio- activity. virus RNA in a cell-free system with special Fractionation of the crude replication complex. emphasis on the possible involvement of termi- The crude replication complex was fractionated by nal adenylate transferase. As we shall outline equilibrium sedimentation in a discontinuous su- in this report, we have used a poliovirus-spe- crose gradient (16). The discontinuous gradient was cific replication complex, first isolated from in- formed by layering the following sucrose-RSB solu- fected HeLa cells by Baltimore (3), which is tions in a 36-ml tube: 3 ml of 60% sucrose in RSB, 7 membrane bound (16, 21) and does not respond ml of 45% sucrose, 7 ml of 40% sucrose, 10 ml of the to exogenous polio RNA template (19). Partial crude replication complex containing 30% sucrose (2 to 3 mg of protein per ml), 7 ml of 25% sucrose in purification of the replication complex was RSB and, lastly, 3 ml of RSB. The gradients were achieved by solubilizing it with deoxycholate centrifuged at 86,000 x g and 4 C for 12 to 17 h in a followed by sucrose gradient centrifugation Spinco SW27 rotor. Caliguiri and Tamm (16) ob- (15). We have found that this complex is free of served seven fractions when the total cytoplasmic detectable poly(A) polymerases or terminal extract was separated by isopycnic banding, the top adenylate transferases, yet it synthesizes fraction (fraction 1) being a lipid layer. In our case, poly(A)-linked polio RNA in vitro. The struc- most of the lipids had been removed during the ture of polio minus-strand RNA, together with preparation of the crude replication complex. The the data presented here, strongly suggests that first visible band in our gradients contains smooth membranes. This fraction has been designated by the genome-linked poly(A) ofpoliovirus is genet- Caliguiri and Tamm (16) as fraction 2 and was found ically coded. to contain poliovirus RNA polymerase at the highest We propose that the biosynthesis of poly(A) specific activity when compared with the other frac- in poliovirus replication occurs by means of tions.